The present invention relates to the change in activity of the cell cAMP signal transduction pathway, which leads to a change in the central cell metabolism and is thus exploited for increasing the production rates for fine chemicals in fermentative processes.
The enzyme adenylate cyclase catalyzes the production of cAMP from ATP and, with the aid of cAMP, governs, in eukaryotes, signal transduction by the enzyme protein kinase A. cAMP binds to the regulatory subunit of protein kinase A, which leads to protein kinase A being activated (Taylor et al., 1990, Annual Rev Biochem: 971-1005). This kinase alters the activity of target proteins by esterifying the hydroxyl groups of specific serines of these proteins with a phosphate group. Protein kinase A is involved in a large number of cellular regulatory processes. On the one hand, the kinase modulates the activity of some transcription factors such as, for example, CREB (Brindle and Montminy, 1992, Curr Opin Genet and Dev, 2, 199-204), which leads to the synthetic rates of metabolic enzymes being altered. Moreover, the product of adenylate cyclase (cAMP) takes part in regulating the central cell metabolism by governing both synthesis and activity of enzymes of glycolysis, gluconeogenesis and the glyoxylate pathway. Thus, cAMP inhibits the synthesis of key enzymes of gluconeogenesis and of the glyoxylate pathway, such as fructose bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase (Boy-Marcotte et al., 1996, Microbiology, 142:459-467). On the other hand, the activity of enzymes of glycolysis, such as phosphofructokinase, is increased by cAMP-mediated protein kinase A activity (Kexcex2ler and Eschrich, 1996, FEBS Lett., 395: 225-227). In addition, the cAMP-activated protein kinase A inactivates the formation of carbohydrate stores in yeasts and stimulates the degradation of carbohydrate stores in fungi (Pall and Robertson, 1988, Biochem Biophys Res Commun, 150: 365-370; Toda et al., 1985, Cell, 40: 27-36). The signal cascade, which is initiated by adenylate cyclase activity, thus plays a crucial role in regulating the substance fluxes in the cell. In particular, cAMP signal transduction alters the direction of the substance fluxes in the central metabolism so that the cAMP signal transduction chain plays a decisive role in providing substrates for synthesizing secondary metabolic products. Specific secondary metabolic products are commercially important fine chemicals. These include, for example, vitamins, carotinoids, amino acids, fragrances and antibiotics.
Fine chemicals have a wide range of industrial uses in animal and human nutrition, in the cosmetics industry and in medicine. In addition, carotinoids act as natural colorants. Fine chemicals are produced industrially by chemical synthesis or, increasingly, by fermentation.
Activation of adenylate cyclase in yeasts and fungi is governed by the RAS G proteins. RAS G proteins here act, for example, as sensors for the glucose concentration in the medium (Field et al., 1990, Science, 247: 464-467). Glucose in the medium leads to RAS being activated by exchanging the bound GDP for GTP. This process is made possible by an RAS-specific exchange factor. The RAS which is activated by GTP binding binds to, and activates, adenylate cyclase, which, in turn, forms cAMP. The signal pathway is switched off by the cAMP-specific phosphodiesterase, which converts cAMP into AMP (Ishikawa et al., 1988, Methods Enzymol., 159, 27-42). Adenylate cyclase is thus a central switchpoint for adaptation to changes in the nutritional environment of cells. In higher eukaryotes, adenylate cyclase is regulated by heterotrimeric G proteins (Federmann et al., 1992, Nature, 356: 159-161).
In prokaryotes, too, adenylate cyclase is involved in the regulation of metabolic processes. Here, cAMP regulates the expression of some catabolic operons by binding to a transcription factor (CRP) (Tagami et al., 1995 Mol Microbiol, 17, 251-258).
To optimize fermentative processes for the production of fine chemicals, the substance flux toward the desired product must be increased.
This can be effected in different ways:
Switching-off of negative regulatory mechanisms of metabolic pathways which lead to the product
Inactivation of enzymes which compete with the enzymes of the metabolic pathways leading to the desired product
Increase, in the cell, of the quantity or activity of enzymes of the metabolic pathways which lead to the product
Since cellular processes proceed in a coordinated fashion owing to higher regulatory mechanisms, such regulatory mechanisms are targets for altering the substance fluxes in the cell.
It is therefore an object to alter regulatory mechanisms in cells in such a way that they allow an increased substance flux toward the desired product.
We have found that this object is achieved according to the invention by altering the activity of the cAMP-dependent signal transduction pathway, which constitutes a central regulator of a large number of metabolic pathways in the cell, so that the substance fluxes can be directed toward the desired product. There is a multiplicity of possibilities of manipulating microorganisms in such a way that they carry an altered activity of the cAMP-dependent signal transduction pathway. However, an altered activity of the cAMP signal transduction pathway for increasing the production of fine chemicals in microorganisms has not been described as yet.
One possibility of increasing the production rates of fine chemicals in microorganisms by altering the activity of the cAMP signal transduction pathway consists in increasing or lowering, in the cell, the enzyme activity of adenylate cyclase, depending on the synthesis to be carried out.
An increase in the quantity of enzyme, and thus an increase in enzyme activity, can be achieved by introducing the gene which encodes adenylate cyclase into the microorganism to be altered at a higher repetition frequency, or by eliminating factors which repress enzyme synthesis. Alternatively, it is possible to exchange the sequences which govern adenylate cyclase expression for sequences which allow increased gene expression. In addition, an increase in enzyme activity can be achieved, for example, by mutating the enzyme to increase substrate conversion, or by disrupting the effect of enzyme inhibitors.
Alternatively, cAMP or a chemical cAMP derivative may be added to the medium, which also leads to protein kinase A being activated.
To reduce the enzyme activity of adenylate cyclase in the cell, the encoding gene can be disrupted, or the activity of adenylate cyclase synthesis activators can be reduced. Adenylate cyclase mutations, which have a reduced activity, can also be applied. Such mutations can be achieved either by traditional methods such as, for example, by UV irradiation or mutagenic chemicals, or directed by means of genetic engineering methods such as deletions, insertions or substitutions.
Not only can the gene activity of adenylate cyclase be altered, it is also possible in the same way to alter the enzyme activity of phosphodiesterase of protein kinase A (catalytic and regulatory domain) and of the RAS proteins (including proteins which have an effect on RAS activity) as described above.
It is preferred to alter the enzyme activity of adenylate cyclase. To reduce the enzyme activity, the adenylate cyclase gene is disrupted. To disrupt the adenylate cyclase gene, a selection marker is inserted into the DNA sequence which encodes adenylate cyclase. Then, this DNA construct is transformed into the cell, where it disrupts the adenylate cyclase gene locus by means of homologous recombination. Thus, the cell is incapable of synthesizing functional adenylate cyclase and thus cAMP.
The invention relates to a gene which contains the nucleotide sequence shown in SEQ ID NO:1 from nucleotide 671 to nucleotide 6295 or a nucleotide sequence which can be obtained therefrom by substitution, insertion or deletion of up to 30%, preferably up to 20%, particularly preferably up to 10%, especially preferably up to 5%, of the nucleotides and, whose gene product has the enzymatic activity of an adenylate cyclase.
The invention furthermore relates to the use of one or more of the abovementioned nucleic acid sequences for constructing, by genetic engineering, microorganisms which compared with the starting organism are capable of increased fine chemical production. A starting organism is to be understood as meaning, in the following text, those microorganisms which are capable of producing the desired fine chemicals, but which carry no genetic alteration with regard to the nucleic acid sequences according to the invention.
The invention is preferably applied to the production of riboflavin.
The preferred nucleic acid sequence used is the adenylate cyclase gene. The adenylate cyclase gene is preferably isolated from the microorganism which synthesizes the fine chemical in question. The preferred microorganisms which are employed in the process according to the invention are fungi and yeasts.
However, it is also possible to use other microorganisms, for example bacteria. Microorganisms which are especially suitable for the production of riboflavin are bacteria of the genus Bacillus and coryneform bacteria of the genus Corynebacteria or Brevibacteria, and yeasts of the genus Candida and Saccharomyces and fungi of the genus Ashbya and Eremothecium. Very particularly suitable for the production of riboflavin are Bacillus subtilis, Corynebacterium ammoniagenes, Candida flareri, Candida famata and Ashbya gossypii. 
The adenylate cyclase gene can be isolated by sequencing a plasmid gene library, by homologous or heterologous complementation of a mutant which carries a defect in the adenylate cyclase gene, or else by heterologous probing or PCR with heterologous primers. For subcloning purposes, the insert of the complementing plasmid can subsequently be minimized in size by suitable cleavage with restriction enzymes. After sequencing and identification of the putative gene, it is fused to the DNA sequence of the selection marker by molecular biology techniques. The plasmid, which carries the sequence of the adenylate cyclase gene, is cleaved in the adenylate cyclase gene, and the selection marker is cloned into this cleavage site. The adenylate cyclase DNA, which frames the selection marker DNA, is subsequently introduced into the cell.
Isolation and sequencing of plasmids from a genomic plasmid library of Ashbya gossypii gives an adenylate cyclase gene which has nucleotide sequences shown in SEQ. ID NO:1 and which, from nucleotide 671 to nucleotide 6295, encode the amino acid sequence given in SEQ ID NO:2 or its allelic variations. Allelic variations encompass, in particular, derivatives which are obtainable from the sequence shown in SEQ.ID NO:1 by means of deletion, insertion or substitution of up to 30%, preferably up to 20%, particularly preferably up to 10%, especially preferably up to 5%, of the nucleotides.
Arranged upstream of the adenylate cyclase genes is, in particular, a promoter of the nucleotide sequence of nucleotide 1-670 as shown in SEQ.ID NO:1 or a sequence which acts essentially the same. Thus, for example, a promoter may be arranged upstream of the gene which differs from the promoter of the abovementioned nucleotide sequence by one or more nucleotide exchanges, by an insertion or insertions, and/or a deletion or deletions, but without the functionality or efficacy of the promoter being adversely affected. Moreover, the promoter may be altered with regard to its efficacy, due to its sequence being altered, or it may be exchanged completely by other promoters.
One or more DNA sequences can be arranged upstream and/or downstream of the adenylate cyclase gene with or without a promoter arranged upstream, or with or without a terminator sequence, so that the gene is retained in its gene structure.
Preference is given to increasing the production of the fine chemical riboflavin (vitamin B2) by altering the cAMP signal transduction pathway. However, other fermentative processes for the production of other fine chemicals are to be optimized by such an alteration. Riboflavin is produced in fungi, yeasts or bacteria, preferably in the fungus Ashbya gossypii. 
If the adenylate cyclase gene in riboflavin-producing microorganism strains of Ashbya gossypii is disrupted, it emerges, surprisingly, that incubation on a glucose-containing medium leads to an increased riboflavin formation. This is seen, inter alia, on culture plates by the visual appearance of the cultures, which show a deep yellow coloration caused by riboflavin, which is weakened by adding cAMP to the medium.